Fig. 2. Anchoring and homomultimerisation with Spy technology. (A) Schematic of unoriented anchoring of a protein to a bead (left) versus oriented SpyTag/SpyCatcher-mediated anchoring (right). Different surfaces bridged using Spy technology are shown. PHA = Polyhydroxyalkanoate. (B) Force spectroscopy using SpyTag anchoring. Sample scheme for directional tethering of a protein of interest (green) between a surface and magnetic tweezers, using SpyTag, HaloTag and spacer domains, to allow repeated folding and unfolding tests. (C) Toolbox for homomultimerisation. SpyAvidins, coiled coils, or protein nanoparticles may be genetically fused to SpyCatcher (marked as a blue dot for ease of visualisation). Addition of SpyTag-fused ligand allows testing of how different multimerisation states change biological effects.